This invention relates to capture of a target entity on a solid phase and detection of the captured target. More particularly, the invention relates to capture and detection of contaminants in biomedical, environmental, and food samples.
Considerable progress in the development of detectors of microbial contamination has been achieved in recent years. These detectors can be applied to medical, process control, and environmental fields. Such detectors must possess features such as high specificity, simplicity, sensitivity, speed, reliability, and reproducibility. S. Y. Rabbany et al., Optical Immunosensors, 22 Crit. Rev. Biomed. Engin. 307-346 (1994). With the use of antibodies as the ligands for specific capture, numerous applications have been developed for detection of pathogenic bacteria. M. R. Blake and B. C. Weimer, Immunomagnetic Detection of Bacillus stearothermophilus Spores in Food and Environmental Samples, 63 J. Appl. Environ. Microbiol. 1643-1646 (1997); A. Burkowski, Rapid Detection of Bacterial Surface Proteins Using an Enzyme-linked Immunosorbent Assay System, 34 J. Biochem. Biophys. Methods 69-71 (1997); S. Chen et al., A Rapid, Sensitive and Automated Method for Detection of Salmonella Species in Foods Using AG-9600 AmpliSensor Analyzer, 83 J. Appl. Microbiol. 314-321 (1997); L. S. Metherell et al., Rapid, Sensitive, Microbial Detection by Gene Amplification Using Restriction Endonuclease Target Sequence, 11 Mol. Cell Probes 297-308 (1997); F. Roth et al., A New Multiantigen Immunoassay for the Quantification of IgG Antibodies to Capsular Polysaccharides of Streptococcus pneumoniae, 176 J. Inf. Dis. 526-529 (1997).
Bacterial spores are the most heat-stable form of microorganisms, are ubiquitous in the environment, and therefore are of great concern in food products, such as milk, that receive extensive heat treatments to prolong shelf life. Spore counts in milk from around the world vary between zero and  greater than 22,000 colony forming units (cfu)/ml depending on the climate of the region. S. Chen, supra. Bacillus stearothermophilus spores are among the most heat-resistant spores and are found in high numbers in soil and water. B. stearothermophilus spores survive extreme heat and will germinate and grow at elevated product storage temperatures, which occur in foods transported in equatorial regions of the world.
While B. stearothermophilus is not commonly a problem, other bacilli often lead to food-borne illness or spoilage in a variety of foods. Bacillus cereus, B. licheniformis, B. subtilis, and B. pumilus, have all been implicated in outbreaks of food-borne illness and are commonly isolated from raw and heat-treated milk. M. W. Griffiths, Foodborne Illness Caused by Bacillus spp. Other than B. cereus and Their Importance to the Dairy Industry, 302 Int. Dairy Fed. Bulletin 3-6 (1995). B. cereus is also responsible for a sweet curdling defect in milk, as well as being pathogenic. W. W. Overcast and K. Atmaram, The Role of B. cereus, in Sweet Curdling of Fluid Milk, 37 J. Milk Food Technol. 233-236 (1973). A mesophilic heat-resistant bacillus similar to Bacillus badius, has been isolated from extreme-temperature-processed milk (D147=5 sec). P. Hammer et al., Pathogenicity Testing of Unknown Mesophilic Heat Resistant Bacilli from UHT-milk, 302 Int. Dairy Fed. Bulletin 56-57 (1995). B. badius is a mesophilic organism that grows readily at room temperature, making it a likely candidate for spoiling temperature-processed foods. There have been 52 confirmed cases of B badius in ultra-high-temperature treated milk in Europe and two cases outside Europe. Lack of a rapid spore assay that can be used in milk contributes to the difficulty of prediction of post-processing spoilage, thereby limiting the shelf life and product safety. H. Hofstra et al., Microbes in Food-processing Technology, 15 FEMS Microbiol. Reviews 175-183 (1994). Such an assay could be used in a hazard critical control point (HACCP) plan allowing raw materials with high spore loads to be diverted to products that do not pose a food safety risk to consumers.
The standard method for quantifying spores in milk, G. H. Richardson, Standard Methods for the Examination of Dairy Products (15th ed., 1985), involves heat-shock and an overnight plate count. This method is time-consuming and merely yields historical information. The food industry needs microbiological assays to yield predictive information for maximum benefit in HACCP analysis and risk assessment. An enzyme-linked immunosorbent assay (ELISA) capable of detecting  greater than 106 cfu/ml of B. cereus spores in food has been reported, but was unacceptable due to antibody cross reactivity. L. A. Metherell et al., supra.
Techniques to increase sensitivity of immunosorbent assays have focused on more efficient reporter labels, such as faster catalyzing reporter-enzymes; signal amplification, such as avidin- or streptavidin-biotin enzyme complexes; and better detectors, such as luminescence and fluorescence. L. J. Kricka, Selected Strategies for Improving Sensitivity and Reliability of Immunoassays, 40 Clin. Chem. 347-357 (1994); W. W. Overcast and K. Atmaram, supra. Immunomagnetic antigen capture is used extensively to separate and identify Escherichia coli and Salmonella from foods. M. R. Blake and B. C. Weimer, supra; S. Y. Rabbany et al., supra; C. Blackburn et al., Separation and Detection of Salmonellae Using Immunomagnetic Particles, 5 Biofouling 143-156 (1991); P. M. Fratamico et al., Rapid Isolation of Escherichia coli O157:H7 from Enrichment Cultures of Foods Using an Immunomagnetic Separation Method, 9 Food Microbiol. 105-113 (1992); A Lund et al., Rapid Isolation of K88+Escherichia coli by using Immunomagnetic Particles, 26 J. Clin. Microbiol. 2572-2575 (1988); L. P. Mansfeild and S. J. Forsythe, Immunomagnetic Separation as an Alternative to Enrichment Broths for Salmonella Detection, 16 Letters Appl. Microbiol. 122-125 (1993); A. J. G. Okrend et al., Isolation of Escherichia coli O157:H7 using O157 Specific Antibody Coated Magnetic Beads, 55 J. Food Prot. 214-217 (1992); Skjerve and Olsvic, Immunomagnetic Separation of Salmonella from Foods, 14 Inter. J. Food Microbiol. 11-18 (1991); D. J. Wright et al., Immunomagnetic Separation as a Sensitive Method for Isolating Escherichia coli O157 from Food Samples, 113 Epidemiol. Infect. 31-39 (1994). These methods, however, involve either a pre-incubation or a subsequent incubation step (usually 18-24hours) to increase the cell numbers for detection. Immunomagnetic capture greatly shortens E. coli and Salmonella testing, but long incubation times limit this method for predictive information. Immunocapture has also been used to quantitate Bacillus anthracis spores in soil samples using luminescence detection, A. Burkowski, supra, but these efforts have led to tests that have a detection limit of about 103 cfu/ml.
In view of the foregoing, it will be appreciated that providing compositions and methods for capture and detection of selected contaminants would be a significant advancement in the art.
It is an object of the present invention to provide compositions and methods of use thereof for capture and detection of contaminants in food, environmental samples, and other applications.
It is another object of the invention to provide compositions and methods of use thereof for capture and detection of contaminants, wherein such methods are highly specific, simple, sensitive, rapid, reliable, and reproducible.
These and other objects can be achieved by providing a composition of matter comprising a ligand component covalently bonded to a nucleic acid component. Preferably, the ligand component is a member selected from the group consisting of antibodies, antigens, lectins, saccharides, and gangliosides, and more preferably is an antibody. In a preferred embodiment, the nucleic acid component is an oligonucleotide. In another preferred embodiment, a linker is interposed between the ligand component and the nucleic acid component. Preferably, the linker is a member selected from the group consisting of polythreonine, polyserine, and dextran, and more preferably is polythreonine. In yet another preferred embodiment, the invention further comprising a label member coupled to the nucleic acid component.
Another composition of matter comprises a solid support covalently bonded to a linker molecule, a nucleic acid component covalently bonded to the linker molecule, and a ligand component covalently bonded to the nucleic acid component. In one illustrative embodiment, the solid support is in the form of a membrane. Preferably, the membrane is a polymer, and more preferably is a member selected from the group consisting of fluorinated polymers, polyolefins, polystyrene, substituted polystyrenes, polysulfones, polyesters, polyacrylates, polycarbonates; vinyl polymers, copolymers of butadiene and styrene, fluorinated ethylene-propylene copolymers, ethylenechlorotrifluoroethylene copolymers, and mixtures thereof. In another preferred embodiment, the ligand component is a member selected from the group consisting of antibodies, antigens, lectins, saccharides, and gangliosides, and preferably is an antibody. Preferably, the nucleic acid component is an oligonucleotide, and the linker is a member selected from the group consisting of polythreonine, polyserine, and dextran, more preferably polythreonine. In another illustrative embodiment, the solid support is in the form of a bead. Preferably, such a bead is a member selected from the group consisting of silicon, glass, silica, quartz, metal oxides, polyvinyl alcohol, polystyrene, poly(acrylic acid), and mixtures thereof.
A method for capturing a target on a solid support comprises:
(a) mixing an aqueous sample containing the target with a first composition comprising a ligand component, configured for binding the target, covalently bonded to a first nucleic acid component such that the ligand component binds the target to result in a complex;
(b) contacting the complex with a second composition comprising (i) a solid support covalently bonded to a linker, (ii) a second nucleic acid component covalently bonded to the linker wherein the second nucleic acid component is complementary to at least a portion of the first nucleic acid component and hybridizes thereto when in contact therewith such that the resulting duplex has a thermal melting temperature of about 60-85xc2x0 C., and (iii) the ligand component covalently bonded to the second nucleic acid component, such that the second nucleic acid component hybridizes to the first nucleic acid component and the ligand component of the second composition binds the target;
(c) heating the aqueous sample to a temperature above the thermal melting temperature of the duplex without denaturing the ligand component and causing the heated aqueous sample to flow by the solid support; and
(d) then reducing the temperature of the aqueous sample to ambient temperature.
A method for detecting a target on a solid support comprises:
(a) mixing an aqueous sample containing the target with a first composition comprising (i) a ligand component, configured for binding the target, covalently bonded to (ii) a first nucleic acid component, and (iii) a label component, such that the ligand component binds the target to result in a complex;
(b) contacting the complex with a second composition comprising (i) a solid support covalently bonded to a linker, (ii) a second nucleic acid component covalently bonded to the linker wherein the second nucleic acid component is complementary to at least a portion of the first nucleic acid component and hybridizes thereto when in contact therewith such that the resulting duplex has a thermal melting temperature of about 60-85xc2x0 C., and (iii) the ligand component covalently bonded to the second nucleic acid component, such that the second nucleic acid component hybridizes to the first nucleic acid component and the ligand component of the second composition binds the target;
(c) heating the aqueous sample to a temperature above the thermal melting temperature of the duplex without denaturing the ligand component and causing the heated aqueous sample to flow by the solid support; and
(d) then reducing the temperature of the aqueous sample to ambient temperature; and
(e) detecting the label component on the solid support.